Polymers of functionalized diaminopropionic acid are efficient mediators of active exogenous enzyme delivery into cells

Delivery of active protein especially enzyme is one of the major therapeutic challenge. Replacing or substituted invalid/improper acting protein offer fast and effective treatment of disease. Herein, we describe the synthesis and properties of biotinylated peptidomimetics consisting of oxoacid—modified 2,3, l-diaminopropionic acid residues with guanidine groups on its side chains. Electrophoretic analysis showed that the obtained compounds interact with FITC-labeled streptavidin or a streptavidin-β-galactosidase hybrid in an efficient manner. Complexes formed by the abovementioned molecules are able to cross the cell membranes of cancer or healthy cells and show promising compatibility with live cells. Analysis of β-galactosidase activity inside the cells revealed surprisingly high levels of active enzyme in complex-treated cells compared to controls. This observation was confirmed by immunochemical studies in which the presence of β-galactosidase was detected in the membrane and vesicles of the cells.


Complex formation and electrophoresis assay
FITC-Strp: Bt-O2Oc-[Dap(GO2)] 6 -O2Oc-NH 2 /Bt-O2Oc-[Dap(GO2)] 8 -O2Oc-NH 2 FITC-streptavidin (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was incubated with increasing concentrations of polymers with N-terminal biotin (compound 1 or 2) or free amino groups (compound 1a or 2a) at molar ratios of 1:1, 1:2, and 1:4 for one hour at RT.Each complex was separated on a native polyacrylamide gel at a concentration of 0.1 or 0.2 µg per lane with polymer and FITC-Strp as controls.A 15-well Native PAGE 4-16%, Bis-Tris, 1.0 mm, Mini Protein Gel (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used.The separation conditions were as follows: 50 mM Bis-Tris, 50 mM tricine, pH 6.8 (running buffer), and constant voltage of 150 V for 2 h at 24 °C.Loading buffer composition: 200 mM Bis-Tris, 6 N HCl, 200 mM NaCl, 40% w/v glycerol, 0.004% bromophenol blue, pH 7.2.Cathode buffer composition: 0.4% Coomassie G-250 diluted 10 times in double distilled water in running buffer.At the final step, the gel was placed in 100 mL of fix solution (40% methanol and 10% acetic acid in water) and microwaved at 1100 watts for 45 s.Next, the gel was shaken for 30 min at room temperature.The fixing solution was removed, and the whole procedure was repeated.After this, the gel was ready for visualization.
The gel was run with reverse polarization at 5 mA initially (45 min); then, after the samples migrated out from the stacking gel, it was run at 20 mA (2 h).Images of the gel were taken using a Fusion Fx (Vilber Lourmat, France) system.
All cells were seeded in 24-well plates (initial number of cells: 10 4 ) and cultured in 0.5 mL of appropriate medium for 24 h.Next, the cells were washed three times with PBS, and 0.5 mL of peptidomimetic (1, 2 or 1a, 2a) in medium was added.The concentration of the all peptidomimetic (1, 2 or 1a, 2a) was 10 µM (molar ratio 1:2) or 20 µM (molar ratio 1:4) respectively.After the indicated time period (2 h, 4 h or 24 h), the cells were washed with PBS and cultured in FluoroBrite DMEM (Gibco, New York, USA).
An ECLIPSE Ti-E inverted fluorescence microscope (Nikon, Tokyo, Japan) was used to evaluate the complex distribution in the cells.The appropriate filters were used for the detection of DAPI and FITC.
All cells were seeded in 96-well plates (initial number of cells: 10 3 ) and cultured in 0.1 mL of appropriate medium for 48 h.Next, the cells were washed with PBS (three times), and 50 µL of appropriate peptidomimetic (1 or 2) in medium was added.The concentration of each peptidomimetic was 10 µM (molar ratio 1:2) or 20 µM (molar ratio 1:4).Cells were cultured for 24 h and then subjected to PBS washes (2 times).After the selected time period, 0 h (t 24h ) or 2 h (t 24h+2h ) (see scheme on Fig. 2) the cells were washed with PBS and lysed with Reporter Lysis Buffer RLB (Promega, Madison, Wisconsin, USA) according to the manufacturer's manual.All lysates were immediately snap frozen at − 80 °C.

Activity studies
The enzymatic activity of β-gal was monitored using resorufin β-d-galactopyranoside (Thermo Fisher Scientific, Waltham, Massachusetts, USA) (final concentration = 2.5 mg/mL dimethyl sulfoxide (DMSO)).The increase in fluorescence was monitored in triplicate using a FluoroStar OMEGA microplate reader (BMG LABTECH, Ortenberg, Germany) with extinction and emission wavelengths of 541 nm and 585 nm. Assay conditions were as follows: 2.5 µL of lysate, 100 µL assay buffer (0.1 M phosphate buffer, pH 7.0) and 5 µL of substrate.The assay was run for 120 min at 37 °C.

In-cell β-gal activity detection
Cells were incubated with the formed complexes for 24 h in culture medium washed by PBS and cultured for additional 2 h or directly subjected to the next step.After this, two washes with PBS were applied to each system; then, the cells were fixed in 3.7% paraformaldehyde in PBS for 15 min.Next, the cells were washed twice in PBS, and 125 µL of reaction mixture was added to each system.The reaction buffer consisted of 40 mM citric acid, 40 mM sodium phosphate, 300 mM NaCl, 10 mM mercaptoethanol, and 4 mM MgCl 2 (pH 6.0) with 2 mM X-Gal (5-bromo-4-chloro-3-indolyl β-d-galactopyranoside) added immediately prior to use from a 34 mM stock in DMSO 17 .The plate was incubated for two hours at 37 °C and observed under an Olympus IX51 microscope www.nature.com/scientificreports/(Olympus, Tokyo, Japan).Application of the X-Gal reagent in the presence of β-gal activity results in a blue, insoluble product that precipitates and can be monitored under an optical microscope.

Cytotoxicity studies
All cell lines were seeded at a concentration of 5 × 10 3 per well in 96-well plates and cultured for 48 h in 100 µL of medium.Next, the medium was replaced with new medium containing the target amount of tested compounds or their complexes.
Each incubation was run in triplicate for 24 h.Untreated cells were used as a control.After 24 h, the medium was removed, and the cells were incubated with MTT-containing medium for 4 h.Next, DMSO was added to each well, and the results were read using a plate reader (SPECTROstar Nano, BMG LABTECH, Ortenberg, Germany) at two different wavelengths, 570 nm and 690 nm.

Immunofluorescence staining and analyses of β-gal in selected cell lines
HB2 or MDA-MB-231 cells were seeded and grown on glass slides (removable 3-well chamber, IBIDI, Gräfelfing, Germany).On the day of the experiment, cells were incubated with a freshly prepared complex of Strp-β-gal and the chosen compound.At the appropriate time points (t 24h and t 24h+2h ) , the cells were washed and subjected to further experimental procedures.
After incubation, the cells were fixed for 10 min in 1% paraformaldehyde in PBS, washed in PBS, permeabilized in 1% Triton X-100 in PBS, and blocked for 4 h with blocking buffer consisting of 2% FBS, 2% bovine serum albumin (BSA) and 0.2% fish gelatin in PBS (lacking Ca 2+ and Mg 2+ ).Next, the cells were incubated for 1 h with β-galactosidase monoclonal primary antibodies (Thermo Fisher Scientific, Waltham, Massachusetts, USA, cat number MA1-152; 1:50), washed four times in 0.1% Tween in PBS followed by DyLight TM 488-conjugated goat anti-mouse IgG secondary antibody diluted in blocking buffer for 45 min (Thermo Fisher Scientific, Waltham, Massachusetts, USA, cat number 35502; 1:50).After 30 min of incubation, Hoechst reagent was added to all systems.After 45 min of incubation, the cells were washed four times in 0.1% Tween in PBS.Blocking solution was used instead of primary antibody as a control for nonspecific staining.Coverslips were mounted on the slides using Fluoromount Aqueous Mounting Medium (Sigma-Aldrich, Darmstadt, Germany).The specimens were imaged with an ECLIPSE Ti-E confocal laser scanning microscope.To determine the mean fluorescence intensity profile of the immunocomplexes, the mean intensity values for green channels was analyzed using NIS-Elements software (Nikon, Tokyo, Japan) with the Automated Measurement Results option.

Statistical analyses
The statistical analyses were performed using GraphPad Prism 8 (Graphpad software, USA).The Shapiro-Wilk test was used to determine normality of the datasets.For data with a normal distribution, the unpaired t-test was used.For other cases, the nonparametric Mann-Whitney test was applied.The results are expressed as mean ± SEM.Values of p ≤ 0.05 were considered statistically significant.

Results
To determine if the biotinylated compounds are able to form complex with protein conjugated streptavidin, we first aimed to confirm the presence of the complex formed by means of polyacrylamide electrophoresis, followed by cytotoxicity study of compounds and formed complexes.The final experiments were designed to provide the information regarding efficacy of transport across the cell membrane of model enzyme.

Synthesis
Two DAPEG polymers consisting of six (Compound 1) or eight (Compound 2) monomers were synthesized and labeled with an N-terminal biotin.Synthesis were performed on solid support using combined synthesis system.Initially DAPEG polymers with 6 or 8 mer length were synthesized using microwave irradiation.After this stage the side chain protecting group were removed and appropriate oxa acid with guanidine group were attached to free amino group of polymer.The molecular weights of the compounds were nearly identical to the calculated weights and ranged from 1947.1 to 2719.3 Da (Table 1).It is generally known that biotin-containing compounds display exceptionally high affinity toward streptavidin or avidin, reaching a subpicomolar dissociation constant.

Complex formation
Next, we aimed to verify the ability of the abovementioned compounds to form a complex with fluorescently labeled streptavidin (FITC-Strp).Electrophoretic separation of a mixture composed of Compound 1 or 1a and www.nature.com/scientificreports/ 2 or 2a with FITC-Strp at three different ratios (1:1, 1:2 or 1:4) resulted in the appearance of additional multiple bands (Fig. 3, boxes in lanes 3-5).In Fig. 4, the same extra bands were observed (boxes in lanes 2-7).www.nature.com/scientificreports/

Cytotoxicity
Our results suggest that the complexes were successfully formed.Next we proceed to determine whether such complexes are able to translocate into the selected cell lines without significant cytotoxicity.Analysis of the cytotoxicity profiles of Compound 1 against a panel of breast cancer cell lines (MDA-MB-231, T47D and SKBR3) and the noncancerous control (HB2) indicated that neither the formed complex nor its constituents significantly impacted cell viability at concentrations up to 20 µM.See Figs. 5 and 6.Based on the above results, we incubated the abovementioned cell lines with FITC-Strp + Compound 1 or 2 in stoichiometric ratios of 1:2 or 1:4 at concentrations (referred to as protein amounts) equal to 10 µM (molar ratio 1:2) or 20 µM (molar ratio 1:4).As shown in Figs. 7, 8, 9 and 10, green fluorescence indicating the presence of FITC-Strp was observed in all cell lines.
No fluorescence was observed for any control system, including FITC alone and FITC-Strp alone.Localization of the signal differed among cell lines and incubation time For HB2, membrane and nuclear localization dominate, whereas for the cancerous cell lines, fluorescence is visible in the cytoplasmic/membrane compartment (Fig. 8).The fluorescence exhibited the highest level of intensity in all systems following a 24-h incubation period, while the efficacy appeared to be insufficient after 2 and 4 h.This observation is pivotal for the purpose of strategizing subsequent experiments, and as a result, we have opted to utilize a 24-h incubation period.The previous reports on CPP utilization indicated that the incubation periods ranged from 1 to 4 h were effective 18,19 .However, it is important to note that this time is highly influenced by the specific cell types employed and other nuanced factors.
Next, we analyzed whether we were able to translocate model proteins with enzymatic activity using these systems, with streptavidin β-galactosidase (Strp-β-gal) conjugate as the selected model.β-Gal is a protein formed by four subunits, each with 1023 amino acid residues, and is frequently utilized as a reporter molecule.The enzyme hydrolyzes lactose and other β-D-galactosides into monosaccharides.Although very specific for the galactose moiety, it tolerates a variety of structures elsewhere in the substrate; thus, any number of organic dyes with a galactose moiety can be used as reporters.For the activity study, we utilized resorufin β-Dgalactopyranoside, which produces free resorufin upon hydrolysis.For the cellular study, X-Gal was used.
To verify the efficacy of complex formation, we performed electrophoretic separation in native polyacrylamide gel with reverse polarization under acidic conditions.This analysis indicated the presence of a complex  The analysis of cytotoxicity against the selected cell lines indicated that HB2 and MDA-MB-231 viability was not significantly influenced by the presence of the tested complexes at concentrations up to 20 µM.Above this concentration, a 20-30% reduction in cell number was observed.For T47D or SKBR3, the complexes significantly reduced the number of live cells at concentrations starting from 10 µM (Fig. 13).These data forced us to exclude T47D and SKBR3 cell lines from further experiments.
Delivery the active enzyme to the cells.Incubation of the formed Strp-β-gal:Bt-O2Oc-[Dap(GO2)] 6/8 -O2Oc-NH 2 complexes with cell lines followed by β-galactosidase activity assays using a fluorescent β-gal substrate (resorufin β-D-galactopyranoside) indicated that after 24 h of incubation, β-gal activity was observed in the majority of the systems, with different activity levels (see Fig. 14).
The greatest fluorescence increase was observed for systems including biotinylated polymers, with increases 2-6-fold greater than those for nonbiotinylated compounds.Culturing the cells for an additional 2 h, resulted in a significant reduction in the activity of β-gal in all systems other than those with complexes formed with biotinylated polymers.The level of recorded enzymatic activity was several times greater than that of the control systems.In MDA-MB-231 cell line the unexpected fluorescence increase is recorded.We are not able to provide reasonable explanations of this cell specific issue.
These observations were confirmed by microscopy observations of intracellular β-gal substrate (X-gal) (Figs. 15, 16); that is, blue color resulting from X-gal enzymatic breakdown was visible on membrane compartment and inside the tested cell lines at all time points, confirming the presence of active enzyme' form.No blue spots were observed for control-nontreated cell lines (Fig. 17).However, some minimal background blue color was observed as the result of endogenous and transported β-gal, since X-gal is a universal substrate for any β-galactosidase activity.
To differentiate between endogenous β-gal activity, which can be found in every living cell at some background level, and the presence of the delivered enzyme, which is the active form of E. coli-expressed β-gal that is present as cargo in this experiment, we performed immunostaining experiments.As shown in Fig. 18, the control systems, cells treated with Strp-β-gal alone or without any additional component, display minimal fluorescence, which indicates the negligible presence of a secondary antibody labeled with DyLight 488 .Significantly elevated fluorescence was observed in all systems in which peptidomimetic Strp-β-gal conjugates were present (Figs.19  and 20).Antibody staining was observed mainly in the membrane compartment but also in vesicle-like structures (Fig. 19b and c).The octamer mimetic was a more effective transporter of the Strp-β-gal complex than the There are several reports describing artificial translocation and subsequent localization of cationic compounds 22,23 .However, in light of all the above described experiments focusing on the localization and activity of the active enzyme form, we are in a position to state that this is not an issue in our case.Analyzing the overall fluorescence observed for particular well, we observed aa pattern that is presented in Fig. 21.Again, the presence of exogenous β-gal after 24 h of incubation with the complexes followed a similar pattern.For the HB2 cell lane, all systems, except the controls, displayed a greater amount of detected β-gal (Fig. 21).MDA-MB-231 cells were more sensitive to the composition and stoichiometry of the complexes used.In first time point (24 h) the greatest fluorescence was observed for compound 1 with dominance a stoichiometric ratio 1:2 system.The compound 2 was less effective in this moment.After additional two hours of incubation the all treated systems with both compounds displayed the same level of fluorescence that indicates effective transport into the cell interior.This observation is in parallel with enzyme activity (Fig. 14) Also microscopic observation recorded after additional 2 h, as visible in Fig. 19 (HB2 cells) and 20 (MDA-MB-231 cells), confirm this observations, the β-gal was mainly visible in vesicle-like structures.Regardless of the different stoichiometric ratios, the efficiency of β-gal in cell delivery remained constant.It is crucial to emphasize that the presence of β-gal as a protein detected by the antibody (Fig. 21) does not always correlate with its level of activity, as demonstrated in Fig. 14.In this particular instance, we observed an absence of direct correlation, but there was a discernible overall trend between the treated and non-treated systems.In summary, the results presented above suggest the efficacy of a novel biotinylated cell-penetrating polymer able to mediate the cellular translocation of a model protein while retaining its activity.Such compounds could be utilized to transport functional exogenous or nonhost proteins into a variety of cells.

Figure 1 .
Figure 1.General scheme of the synthesized compounds.

Figure 2 .
Figure 2. Incubation scheme for Strp-β-gal:peptidomimetics.Additional two hours of culturing allow the membrane bound complex to dissociate.

Figure 14 .
Figure 14.Activity of β-gal in cell lysates of HB2 or MDA-MB-231 cells incubated for 24 h with the indicated compound or mixtures (time point t 24h ) and 24 h followed by 2 h of culture in fresh medium (time point t 24h + 2h ) (mean ± SD; n = 3).Statistical calculations were performed using one-way ANOVA: ****p < 0.0001.

Figure 21 .
Figure 21.Mean fluorescence intensity of secondary antibody goat anti-mouse IgG DyLight TM 488 conjugates in various systems.